Bones, along with a small number of cartilages, comprise the skeletal system that serves as the rigid supporting framework of the body in adult humans. Certain parts of the supporting framework form chambers, such as the skull and the thoracic cage, that are important for protecting the soft parts contained in the chambers. Bones also serve as attachments for muscles and act as levers in the joint system of the body.
Mature bone is comprised of an organic framework of fibrous tissue and inorganic salts known as crystalline hydroxyapatite (HA). HA is composed of calcium and phosphorous, which are derived from the blood plasma and ultimately from nutritional sources. HA represents about 60 percent of the weight of compact bone and is deposited on a fibrous structure of collagenous connective tissue. Without HA, bone loses most its weight and rigidity and is susceptible to damage.
The process of bone formation, also known as osteogenesis, involves three main steps: production of the extracellular organic matrix (osteoid); mineralization of the matrix to form bone; and bone remodeling by resorption and reformation. The cellular activities of osteoblasts, osteocytes, and osteoclasts are essential to the process.
Osteoblasts synthesize the collagenous precursors of bone matrix and also regulate its mineralization. During bone formation, osteoblasts line tiny spaces known as lacunae within the surrounding mineralized matrix. Osteoblasts that line the lacunae are called osteocytes. Osteocytes occupy minute canals (canaliculi) which permit the circulation of tissue fluids. Hormones, growth factors, physical activity, and other stimuli act mainly through osteoblasts to bring about effects on bone. Osteoclasts are derived from hematopoietic stem cells that also give rise to monocytes and macrophages. Osteoclasts adhere to the surface of bone undergoing resorption and lie in depressions referred to as resorption bays. Osteoclasts are apparently activated by signals from osteoblasts. Osteoclastic bone resorption does not occur in the absence of osteoblasts. To meet the requirements of skeletal growth and mechanical function, bone undergoes dynamic remodeling by a coupled process of bone resorption by osteoclasts and reformation by osteoblasts.
Bone is formed through one of two pathways, by replacement of cartilage or by direct elaboration from periosteum. These processes are known, respectively, as endochondral ossification and intramembranous ossification. During endochondral ossification, a cartilaginous bone model is first formed. Then, a layer of bone on the surface of the cartilaginous shaft is formed by osteoblasts. Succeeding layers of bone follow. At the same time, the matrix of cartilage cells is calcified into a trabecular network of cartilage while the interstitial cartilage is degenerated. The combined processes of calcification and degeneration of the cartilage advance from the center toward the ends of the cartilage model. The osteoblasts penetrate the cartilage along with capillaries to produce bone on the cartilaginous trabeculae and advance from the center to the ends to progressively form bone on the cartilaginous trabeculae. Ultimately, the calcified cartilage is completely replaced by spongy bone.
In contrast, the process of intramembranous ossification does not involve a cartilaginous template. Instead, mesenchymal cells become osteoblasts which begin to form the branching trabeculae of bone. The initial thin trabeculae are some times referred to as spicules. The trabecular bone becomes denser by widening of the trabeculae, and is then remodeled externally and internally. The mandibles, clavicles and certain bones of the skull are produced through intramembranous ossification.
There are a number of diseases related to bone formation, deterioration and healing, including osteoporosis, osteogenesis imperfecta (OI) and fibrodysplasia ossificans progressiva (FOP). Osteoporosis, or porous bone, is a disease characterized by low bone mass and structural deterioration of bone tissue, leading to bone fragility and an increased susceptibility to fractures of the hip, spine, and wrist. Osteoporosis is a major public health threat for more than 28 million Americans, 80 percent of whom are women. The strength of bone depends on its mass and density. Bone density depends in part on the amount of calcium, phosphorus and other minerals bones contain. Bones that contain less mineral are weakened and lose internal supporting structure. A full cycle of bone remodeling takes about 2 to 3 months. Children tend to make new bone faster than old bone is broken down. As a result, bone mass increases. Peak bone mass is reached in an individual's mid-30s. Although bone remodeling continues, old bone is broken down faster than new bone is formed. As a result, adults lose slightly more bone than is gained—about 0.3 percent to 0.5 percent a year. Lack of vitamin D and calcium in an individual's diet can accelerate the process. In addition, for women at menopause, estrogen levels drop and bone loss accelerates to about 1 percent to 3 percent a year. Bone loss slows but doesn't stop at around age 60. Women may lose between 35 percent and 50 percent of their bone mass, while men may lose 20 percent to 35 percent of their bone mass. Development of osteoporosis depends on the bone mass attained between ages 25 and 35 (peak bone mass) and how rapidly it is lost as an individual get older. The higher an individual's peak bone mass, the less likely that individual will develop osteoporosis. Calcium, vitamin D and exercising regularly are important for maintaining bone strength. Nonetheless, methods for effectively treating osteoporosis are still desired.
Osteogenesis Imperfecta (OI) is a genetic disorder characterized by bones that break easily, often from little or no apparent cause. There are at least four distinct forms of the disorder, representing extreme variation in severity from one individual to another. For example, a person may have as few as ten or as many as several hundred fractures in a lifetime. While the number of persons affected with OI in the United States is unknown, the best estimate suggests a minimum of 20,000 and possibly as many as 50,000. OI can be dominantly or recessively inherited and can also occur as a mutation. A cure for OI has not yet been discovered. As a result, methods for treatment focus on preventing and controlling symptoms, strengthening bone mass and ensuring proper healing.
In addition, both osteoporosis and OI leave patients vulnerable to bone fractures. If these bone fractures do not heal properly, these patients may continue to suffer from pain and may be at increased risk for further fractures as well as other related complications. Methods or treatments that enhance and/or ensure proper fracture healing are important for patients with osteoporosis or brittle bone disease. Another group that will benefit from methods for enhanced wound healing are the elderly and patients who have undergone orthopaedic procedures.
Fracture healing is the culmination of a highly orchestrated series of physiological and cellular pathways to restore the function of broken bones. Fracture healing generally involves the following steps: the formation of a hematoma (collection of blood at the fracture site), development of a soft callus due to cell multiplication in the lining of the injured bone, growth of blood vessels and fibrocartilege in the middle of the fracture, formation of osteoblasts that migrate into the callus and deposit calcium to form a hard callus, and remodeling and strengthening of the bone through osteoblast and osteoclast formation.
Osteogenesis during fracture healing occurs by intramembraneous and endochondral ossification that histologically resembles fetal skeletogenesis (Einhorn 1998; Vortkamp et al. 1998; Ferguson et al. 1999). However, the localized tissue hypoxia, the fracture hematoma, subsequent inflammation at the fracture site, and the frank remodeling of the fracture callus at the later stages of healing are unique physiological and cellular responses to bone fractures that have no known corresponding counterpart during fetal development of the skeleton.
It has been hypothesized that the early physiological responses to a bone fracture, namely hypoxia and inflammation, induce gene expression pathways and promote cell proliferation and migration into the fracture site in order to promote healing (Brighton et al. 1991; Bolander 1992). Production or release of specific growth factors, cytokines, and local hormones at the fracture site by these physiological processes would create the appropriate microenvironment to (1) stimulate periosteal osteoblast proliferation and intramembraneous ossification to form the hard fracture callus, (2) stimulate cell proliferation and migration into the fracture site to form the soft callus, and (3) stimulate chondrocyte differentiation in the soft callus with subsequent endochondral ossification. Remodeling of the fracture callus by osteoclastic resorption and subsequent osteogenesis converts the fracture callus woven bone into cortical bone and thereby restores the shape and mechanical integrity of the fractured bone.
One potential class of factors that would mediate certain events of fracture healing is the prostaglandins. The effects of prostaglandins on bone metabolism are complex since prostaglandins can stimulate bone formation as well as bone resorption (Kawaguchi et al. 1995). However, because the in vivo half-life of purified or synthetic prostaglandins is very short, prostaglandins per se have a limited therapeutic value.
Prostaglandins are synthesized by osteoblasts and different cell stimuli can alter the amount and possibly the spectrum of prostaglandins produced by osteoblasts (Feyen et al. 1984; Klein-Nulend et al. 1997; Wadleigh and Herschman 1999). Therefore, signal transduction, mechanical perturbations, or other physiological signals can affect bone metabolism through altercation of prostaglandin production.
Prostaglandin synthesis begins with the release of arachidonic acid from membrane phospholipids by phospholipase activity. Arachidonic acid is subsequently converted into prostaglandin H2 (PGH2) by cyclooxygenase (COX) via two independent catalytic steps (Needleman et al. 1986). Synthase enzymes then convert PGH2 into the specific prostaglandins produced by that cell such as PGD2, PGE2, PGF2α, prostacyclin, and thromboxane. Thus, cyclooxygenase activity is essential for normal prostaglandin production and cyclooxygenase is believed to be the rate-limiting enzyme in the prostaglandin synthetic pathway.
There are two known forms of cyclooxygenase, COX-1 and COX-2, which are encoded by two genes (Xie et al. 1991; O'Banion et al. 1992). COX-1 is constitutively expressed by many tissues and provides a homeostatic level of prostaglandins for the body and specific organs, such as the stomach and kidneys (Vane et al. 1998). In contrast, COX-2 is inductively expressed in vitro by a diverse array of cell stimuli such as exposure to lipopolysaccharide (O'Sullivan et al. 1992a; O'Sullivan et al. 1992b), certain cytokines and growth factors (O'Banion et al. 1992; Wadleigh and Herschman 1999), or mechanical stress (Topper et al. 1996; Klein-Nulend et al. 1997). COX-2 expression can be stimulated in vivo by wounding and inflammation (Masferrer et al. 1994; Shigeta et al. 1998; Muscaráet al. 2000).
Inhibiting the cyclooxygenase activity of COX-1 and COX-2 can reduce prostaglandin synthesis by preventing the conversion of arachidonic acid into PGG2, the precursor of PGH2. This is commonly done to reduce inflammation and pain with aspirin and non-steroidal anti-inflammatory drugs (NSAIDs), such as indomethacin. Most NSAIDs inhibit the cyclooxygenase activity of COX-1 and COX-2 with near equal potency, which often leads to detrimental gastro-intestinal or kidney side effects (Raskin 1999; Whelton 1999). Use of COX-2-selective NSAIDs has become very popular since these drugs, such as celecoxib (Celebrex) and rofecoxib (Vioxx) preferentially inhibit the cyclooxygenase activity of COX-2 with selectivity relative to COX-1 of approximately 8-fold for celecoxib and 35-fold for rofecoxib (Riendeau et al. 2001).
Prostaglandins are produced during fracture healing. Prostaglandin levels in and around the healing callus of rabbit tibia that had been severed by osteotomy showed that PGE and PGF levels were elevated between 1 and 14 and 7 and 14 days post-osteotomy, respectively (Dekel et al. 1981). No survey of the temporal pattern or variety of prostaglandins produced during fracture healing has been reported for other rodents or man. Non-specific NSAIDs have been shown to delay but not stop fracture healing in experimental animal models (Rø et al. 1976; Allen et al. 1980; Altman et al. 1995). In addition, non-specific NSAIDs have been shown to reduce the incidence and severity of heterotopic (abnormal or deviating from the natural position) bone formation in humans following certain fractures or orthopaedic surgical procedures (Pritchett 1995; Moore et al. 1998). These observation suggest that prostaglandins are necessary for bone formation but given the limitations of non-specific NSAID use, it is unknown whether prostaglandins produced by COX-1, COX-2 or both enzymes are essential for fracture healing.